On the Origins of Homology Directed Repair in Mammalian Cells

International Journal of Molecular Sciences

Editorial On the Origins of Homology Directed Repair in Mammalian Cells

Brett M. Sansbury and Eric B. Kmiec *

Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, ChristianaCare, Newark, DE 19713, USA; [email protected] * Correspondence: [email protected]

Abstract: Over the course of the last ﬁve years, expectations surrounding our capacity to selectively modify the human genome have never been higher. The reduction to practice site-speciﬁc nucleases designed to cleave at a unique site within the DNA is now centerstage in the development of effective molecular therapies. Once viewed as being impossible, this technology now has great potential and, while cellular and molecular barriers persist to clinical implementations, there is little doubt that these barriers will be crossed, and human beings will soon be treated with gene editing tools. The most ambitious of these desires is the correction of genetic mutations resident within the human genome that are responsible for oncogenesis and a wide range of inherited diseases. The process by which gene editing activity could act to reverse these mutations to wild-type and restore normal protein function has been generally categorized as homology directed repair. This is a catch-all basket term that includes the insertion of short fragments of DNA, the replacement of long fragments of DNA, and the surgical exchange of single bases in the correction of point mutations. The foundation of homology directed repair lies in pioneering work that unravel the mystery surrounding genetic exchange using single-stranded DNA oligonucleotides as the sole gene editing agent. Single agent gene editing has provided guidance on how to build combinatorial approaches to human gene editing using the remarkable programmable nuclease complexes known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their closely associated (Cas) nucleases. In this manuscript, we outline the historical pathway that has helped evolve the current molecular toolbox Citation: Sansbury, B.M.; Kmiec, E.B. being utilized for the genetic re-engineering of the human genome. On the Origins of Homology Directed Repair in Mammalian Cells. Int. J. Keywords: homology directed repair; gene editing; CRISPR-Cas; CRISPR Mol. Sci. 2021, 22, 3348. https://doi. org/10.3390/ijms22073348

Received: 23 February 2021 Accepted: 22 March 2021 1. Introduction Published: 25 March 2021 Long before the advent of programmable nucleases, the field of gene editing was advancing methodically toward elucidating the molecular mechanism by which mam- Publisher’s Note: MDPI stays neutral malian genes can be manipulated specifically by exogenously added reagents. In almost with regard to jurisdictional claims in all cases, site-specific changes were executed by single-stranded DNA oligonucleotides published maps and institutional affil- (ssODNs) that penetrated the nuclear membrane and invaded the double helix providing iations. the information to facilitate the desired nucleotide exchange. These early studies provided guidance toward understanding how genomic changes are executed [1,2]. As gene editing technologies advance, recent studies centered on the combinatorial action of ssODNs and programmable nucleases, such as CRISPR-Cas complexes, have continued along this line Copyright: © 2021 by the authors. of investigation, elucidating our understanding of how eukaryotic genes can be genetically Licensee MDPI, Basel, Switzerland. re-engineered. In this manuscript, we define the origins and the evolution of the gene This article is an open access article editing reaction with a specific focus on homology directed repair. distributed under the terms and conditions of the Creative Commons 2. Improving Efficiency by Modifying the Donor DNA Attribution (CC BY) license (https:// The gene editing strategy utilizing the simple addition of single-stranded DNA tem- creativecommons.org/licenses/by/ plates, known as single agent gene editing, takes place in a two-phase reaction, pairing 4.0/).

Int. J. Mol. Sci. 2021, 22, 3348. https://doi.org/10.3390/ijms22073348 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 3348 2 of 6

and repairing [3]. At its most fundamental level, the oligonucleotide is designed to pair with its complementary region within the target gene apart from a central base purposely constructed to create a mismatch (pairing). The endogenous DNA repair systems recog- nize the artificial mismatch and direct the resolution of the mis-paired base (repairing). Thus, gene repair or nucleotide exchange is facilitated by the natural action of biological pathways inherent in the cell. Seminal studies conducted in E. coli and Saccharomyces cerevisiae established the validity of synthetic DNA, ssODNs, as a donor template to direct site-specific base changes. Mandecki et al. [4] demonstrated permanent changes in plasmid DNA using oligonucleotides transformed into bacteria. Shortly thereafter, Sherman and colleagues published a series of important papers in which ssODNs were introduced into Saccharomyces cerevisiae with the goal of making inheritable changes in genes that determine the auxotrophic status of the organism [5]. Yammamoto et al. [6] targeted the CYC1 gene and described a distinct strand bias or polarity in the mutagenesis of gene editing reactions. Thus, foun- dational studies helped reveal the potential for using a simple strategy for making single base changes in chromosomal DNA. The first transition to mammalian cells came from the work of Campbell et al. [7] who reported successful editing of an episomal target. The frequency with which gene repair took place was low, requiring a certain level of selection to identify cells that had undergone some degree of genetic alteration. Selection protocols were less effective in mammalian cells as the choice of reliable selection agents and protocols were limited, since they continue to be today. Thus, a more complete understanding of the rate limiting steps of gene editing was needed to provide information as to how the frequency of conversion in mammalian cells could be elevated. The mechanism of action investigations into homologous pairing and strand exchange reactions, most often catalyzed by the RecA protein, revealed that the alignment of complementary stands into homologous register was a rate limiting step in the overall process [8,9]. Regardless of how the target site becomes available for pairing, the invasion of the single- stranded DNA into the duplex creates a key intermediate structure, the so-called D- loop, known as a three-stranded structure with a dynamic flux of unstable and stable base pairing in the region of complementarity (Figure1). Once stabilized, the D-loop can be resolved in a variety of ways, leading to successful homologous exchange or disassembly of the pairing partners without genetic change. Importantly, the stability and half-life of the D-loop appears to be the governing factor in determining the outcome of homologous pairing. This remains true in CRISPR-directed gene editing since a series of molecular processes including replication, repair, and recombination take place within the interwoven complex of strands present at the invasion site. Early efforts to increase stability of the three-stranded intermediate included the synthesis of triplex forming oligonucleotides (TFOs) and bifunctional oligonucleotides [10,11] and even DNA fragments tethered to TFOs [12]. Chemical and structural modifications showed some improvement including work by Ali-McNeer et al. [13] who tethered the TFO to a donor DNA strand. This complex was designed to repair the well-known F508 deletion mutation in Cystic Fibrosis. Significant correction of this mutation was achieved by creating a correction tool in which the targeting molecule was essentially constructed as a molecular clamp, positioning the active part to execute gene editing. In an alternative approach, RNA was incorporated directly into the single-stranded DNA oligonucleotide to create a chimeric oligonucleotide, which, by its very nature, elevated hybridization to the complementary DNA sequence at or near the target site and improved the targeting efficiency [9,14,15]. Then, in 2004, Drury et al. [16] provided confirmatory evidence that the D-loop is, in fact, the critical reaction intermediate in gene repair. Taken together, this work supported the concept that improving the stability of gene editing reaction intermediates could improve the efficiency with which overall gene editing or repair took place. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 7

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Figure 1. The D-Loop. A three stranded reaction intermediate is created when a single-stranded DNA Figure 1. The D-Loop. A three stranded reaction intermediate is created when a single-stranded DNAmolecule molecule serving serving as the as donor the donor strand strand for homology for homology directed directed repair interacts repair interacts with this with complementary this com- plementarystrand within strand the helix. within This the step helix comprises. This step the comprises ﬁrst phase the of first genetic phase engineering of genetic engineering and DNA pairing. and DNAThe dynamic pairing. bindingThe dynamic of the binding single-stranded of the single DNA-stranded with its complementDNA with its creates complement the structure creates known the structureas the D-loop. known If as a the single D-loop. nucleotide If a single base nucleotide is engineered base intois engineered the donor into strand the donor so that strand it creates so a thatmismatch it creates with a mismatch the nucleotide with inthe the nucleotide helix. Natural in the cellular helix. Natural DNA repair cellular enzymes DNA repair should enzymes then act to shouldcorrect then the mismatch act to correct and the convert mismatch a mutant and conv baseert to aa normalmutant base.base to This a normal step comprises base. This the step second comprisesphase of gene the second editing phase known of as gene DNA editing repairing. known as DNA repairing.

3. EnhancingEarly efforts the to Frequency increase stability of Single of Agentthe three-stranded Gene Editing intermediate by Changing included the the syn- thesisCellular of triplex Environment forming oligonucleotides (TFOs) and bifunctional oligonucleotides [10,11] and evenOne concernDNA fragments about extensive tethered chemical to TFOs modifications[12]. Chemical to and the donorstructural template modifications was that showedthey could some lead improvement to an immune including response. work Thus, by while Ali-McNeer some workers et al. [13] continued who tethered to focus the on TFOthe modification to a donor DNA of the strand. donor, otherThis complex groups began was designed to circle around to repair the the idea well-known of modifying F508 the deletioncellular environmentmutation in Cystic within Fibrosis. which gene Significant editing took correction place. Byof this far, themutation most productive was achieved and bysuccessful creating strategya correction was thetool modulationin which the of targeting the cell cycle molecule [1,17 ].was Whereas essentially normal constructed reactions asusing a molecular single-stranded clamp, positioning DNA templates the active under part standard to execute cell gene growth editing. conditions In an alternative hovered approach,between 0.3% RNA and was 1% incorporated at best, it was directly found into that, the if single-stranded the donor templates DNA were oligonucleotide introduced tointo create the cellsa chimeric during oligonucleotide, their transition throughwhich, by the its S very phase, nature, the frequencies elevated hybridization of gene editing to theincreased complementary three-fold DNA to five-fold. sequence Increasing at or near the the populations target site and of cellsimproved in the the S phase targeting was efficiencyachieved using[9,14,15]. cell Then, synchronization in 2004, Drury with et the al. population[16] provided accumulating confirmatory primarily evidence at that the theG1/S D-loop border, is, in followed fact, the by critical release reaction into the intermediate S phase en masse. in gene If repair. transfection Taken occurredtogether,after this workrelease, supported the DNA the replication concept that activity improving was slowed the stability as the cellsof gene sensed editing an increasedreaction interme- amount diatesof DNA could ends. improve This stalling the efficiency engages with the DNAwhich damage overall gene response editing pathway or repair and took stimulates place. enzymatic activities required for gene editing [18]. The transitory slowdown of replication 3.forks, Enhancing in turn, the enabled Frequency an even of Single more Agent proficient Gene penetration Editing by of Changing the chromatin the Cellular structure Environmentby the single-stranded donor, once again enhancing target accessibility and improving gene editing frequency. The same sort of effect could also be obtained by incorporation of One concern about extensive chemical modifications to the donor template was that dideoxy compounds such as ddC, which would, in effect, slow the rate of DNA synthesis they could lead to an immune response. Thus, while some workers continued to focus on by reducing replication fork movement [19]. the modification of the donor, other groups began to circle around the idea of modifying These discoveries have held true for elevating gene editing using CRISPR-Cas [20,21]. the cellular environment within which gene editing took place. By far, the most produc- Lin et al. [22] demonstrated the same type of dependency using a Cas9 ribonucleoprotein tivecomplex and successful in primary strategy neonatal was fibroblasts the modulati as wellon of as the human cell cycle embryonic [1,17]. stemWhereas cells. normal Thus, reactionsthe relationship using establishedsingle-stranded between DNA gene templates editing andunder cell standard cycle progression cell growth for singleconditions agent hoveredactivity has between remained 0.3% applicable and 1% toat combinatorialbest, it was found gene editingthat, if usingthe donor programmable templates nucleases.were introduced into the cells during their transition through the S phase, the frequencies of gene editing increased three-fold to five-fold. Increasing the populations of cells in the S phase was achieved using cell synchronization with the population accumulating primarily at

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4. Enhancing the Frequency of Single Agent Gene Editing Using Double-Strand DNA Breakage Double-stranded breaks in chromosomes set in motion a cascade of events that lead to successful repair of the break, mutagenesis, or loss of cell viability. The repair of broken chromosomal DNA most often occurs by non-homologous end joining (NHEJ), which is a process where the broken ends of the DNA duplex are ligated back together to rescue the integrity of the chromosome and prevent mis-segregation at mitosis. Alternatively, the breaks in the chromosome can be restored by the process of homologous recombination, most often occurring in meiosis, and requiring a DNA repair template that bears some level of complementarity. The canonical pathway of NHEJ is operational through each phase of the cell cycle and is most likely the preferred pathway when chromosome integrity is in jeopardy. It requires far less energy [23–25]. Such activity serves to maintain accurate and balanced DNA content. A second type of non-homologous end joining is referred to as alternative NHEJ (alt-NHEJ), which is a process where double-strand breaks un- dergo a limited amount of resection and repair via micro-homology mediated end joining (MMEJ) [26,27]. While survival of the cell is the goal of such conjoining, NHEJ can be mutagenic, introducing insertions and/or deletions (indels) and breakpoint abnormalities. In contrast, homologous recombination maintains and even increases genetic diversity while ensuring genome integrity. Since double-strand DNA breaks can initiate genome rearrangement, and, with the emergence of programmable nucleases able to cleave at a specific site, the natural pathways of homologous recombination have become the foundation for the new age of genome engineering. Workers studying single agent gene editing recognized that a double-strand break can also slow the progression of the cell cycle, impacting cells particularly transiting the S phase [28]. Thus, the generation of free ends ultimately consider recombinogenic can, in some cases, lead to the retardation of cells moving through S and G2 [18,19,29]. Under these conditions and in response to DNA damage, cell can once again become amenable to modification. Prior to the development of programmable nucleases, workers in the field used anticancer drugs such as VP16, bleomycin, or camptothecin to induce the double-strand breaks. The advantage of this approach is that a seamless delivery into the nucleus takes place since these drugs are small enough to penetrate the nuclear membrane without active transport. The downside is that multiple breaks occur at random within the chromosomal array, raising the possibility that unwanted and unwarranted mutagenesis could take place. Site-specific nuclease activity promoted by CRISPR-Cas systems have reduced the propensity for off-site cleavage because they can be designed to target and initiate a break site within the genome at the site designated for the change. This advance alone has been transformative for gene editing in mammalian cells.

5. The Cellular Cost of Genome Engineering The delay in cell cycle progression has been termed reduced proliferation phenotype [30] (Figure2). This phenotype emerges from observations in which the actual percentage of corrected cells within a population diminishes over time due, in large part, to the continual outgrowth of uncorrected cells. These cells are not adversely affected by the introduction of the single-stranded template, modiﬁed or not. The gross of the uncorrected cells reduces the percentage of cells that have been effectively edited through the process of simple dilution. The fact that only corrected cells exhibit this reduced proliferation phenotype leads to the conclusion that the chemical modiﬁcation of single-stranded DNA template is not the primary cause for DNA damage activation of the slowdown in cell progression. It may be as simple as high levels of oligonucleotide, often required to direct effective nucleotide exchange in single agent gene editing, activate the DNA damage response, and subsequent reduction cell proliferation. Conversely, cells that have not received as much oligonucleotide remain unimpeded in terms of replication activity and, therefore, maintain a consistent level of proliferation. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 7

6. The Cellular Cost of Genome Engineering The delay in cell cycle progression has been termed reduced proliferation phenotype [30] (Figure 2). This phenotype emerges from observations in which the actual percentage of corrected cells within a population diminishes over time due, in large part, to the continual outgrowth of uncorrected cells. These cells are not adversely affected by the introduction of the single-stranded template, modified or not. The gross of the uncorrected cells reduces the percentage of cells that have been effectively edited through the process of simple dilution. The fact that only corrected cells exhibit this reduced proliferation phenotype leads to the conclusion that the chemical modification of single-stranded DNA template is not the primary cause for DNA damage activation of the slowdown in cell progression. It may be as simple as high levels of oligonucleotide, often required to direct effective nucleotide exchange in single agent gene editing, activate the DNA damage response, and subsequent reduction cell proliferation. Conversely, cells that have not re- Int. J. Mol. Sci. 2021, 22, 3348 ceived as much oligonucleotide remain unimpeded in terms of replication activity and,5 of 6 therefore, maintain a consistent level of proliferation.

FigureFigure 2. Regulation 2. Regulation of gene of geneediting editing in mammalian in mammalian cells. cells.In this In diagram, this diagram, traditional traditional pairing pairing and and repairingrepairing (or resolution) (or resolution) phases phases of gene of gene editing editing are expanded are expanded to include to include specific speciﬁc reactions reactions that thattake take placeplace before, before, during, during, and andafter after nucleotide nucleotide exchan exchange.ge. Importantly, Importantly, a whole a whole series series of metabolic of metabolic events events including DNA replication and DNA repair are active. including DNA replication and DNA repair are active.

7. Conclusions6. Conclusions The TheNobel Nobel laureate laureate Arthur Arthur Kornberg Kornberg once once said said that that it is it a is crime a crime to towaste waste clean clean think- thinking ing on dirty enzymes. His logic was basedbased onon thethe largelylargely heldheld tenettenet that,that,until until we we understand under- standthe the activities activities of of the the fundamental fundamental components components of of a moleculara molecular reaction, reaction, using using purified purified DNA DNAtemplates templates and and purified purified enzymes, enzymes, we we shall shall never never truly truly appreciate appreciate how how best best to to improve im- provetheir their collective collective activities. activities. Early Early purveyors purveyors of gene of gene editing editing followed followed this advicethis advice by utilizing by utilizingpurified purified donor donor DNA DNA templates templates in the in the absence absence of otherof other biomolecules. biomolecules. Single Single agent agent gene geneediting editing has has been been useful useful in mechanisticin mechanistic studies studies aimed aimed at elucidating at elucidating the regulatorythe regulatory circuitry circuitrysurrounding surrounding gene editinggene editing in mammalian in mammalian cells. Observations cells. Observations from studies from in studies which single-in whichstranded single-stranded DNA oligonucleotides DNA oligonucleotides were the solewere genetic the sole tool genetic have tool provided have foundationalprovided foundationalinformation information and important and guidanceimportant to guid improveance safeto improve and efficacious safe and genome efficacious modification. ge- nomeChemical modification. and structural Chemical modifications and structural of modifications the donor template of the improve donor template stability. improve Some of the stability.most Some important of the observations most important center observations on the concept center that on the concept cell must that be the viewed, cell must in large be viewed,part, as in a livinglarge part, organism. as a living Efforts organism to engineer. Efforts the to sophisticated engineer the tools sophisticated to reacha tools specific outcome can have long-term and potentially mutagenic effects on the functionality and proliferation of the cells we aim to change. It will be interesting to see if we take advantage of what history has taught us.

Author Contributions: B.M.S. and E.B.K. wrote and edited the manuscript. Both authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Gene Editing Institute (GEI) Endowment Fund, generously provided by the Ammon Foundation (AP-130). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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